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Low Concentration and High
Transparency Keratin Hydrogel
Fabricated via Cryoablation
Xiaoqing Wang
1
,
2
, Zhiming Shi
1
*, Le Zhao
2
and Xianyi Shen
2
1
School of Materials Science and Engineering, Inner Mongolia University of Technology, Hohhot, China,
2
College of Textile and
Light Industry, Inner Mongolia University of Technology, Hohhot, China
Keratins are highly attractive for medical applications due to their inherent self-assemblies
characteristics and biocompatibility. However, nearly all researches have focused on the
properties of hybrid hydrogels which was prepared from human hair keratin with other
materials, and the preparation methods and properties of pure keratin hydrogels are rarely
studied. Thus, we extracted keratins from rabbit hair, and a low concentration and high
purity RHK hydrogel was then prepared by a simple freeze–thaw cycle and used to study
gelation and the optical properties. The results indicated that RHK keratin hydrogel is a
reversible thixotropic system and elastic modulus the storage modulus (G′) substantially
improves with freeze–thaw cycles. The systematic assessments including microstructural
observation, porosity, and the secondary structure confirmed that the structure and
properties of keratin hydrogels can be changed by controlling freeze–thaw cycles.
Meanwhile, it is found that RHK hydrogel had high optical transmittance, and still
maintained its fluorescent properties, which would be useful to observe the wound
healing and locate the drug delivery process.
Keywords: keratin hydrogels, freeze–thaw, rheology, morphology, optical properties
INTRODUCTION
As a renewable resource, keratin resources are very abundant, it is found widely in human and animal
organs, including epidermis, hoof, horn, hairs, feather, and protein fiber (Shavandi et al., 2017). For a
long time, people have been working on extracting keratin from fiber, studying its biocompatibility
and degradability for tissue engineering and medical applications (Esparza et al., 2018a), such as
wound healing (Wang et al., 2017;Kim et al., 2019;Konop et al., 2020), hemostatic dressings (Sun
et al., 2018;Tang et al., 2021), controlled drug delivery (Guo et al., 2014;Nakata et al., 2015;Sun et al.,
2016), and antibacterial wound dressing (Zhai et al., 2018;Sadeghi et al., 2020). Using a riboflavin-
SPS-hydroquinone (initiator–catalyst–inhibitor) photosensitive solution, Placone and others
prepared keratin scaffolds through 3D printing technology (Placone et al., 2017). Despite these
advantages, these biomaterials are mainly prepared from keratin mixed with other materials because
pure keratin template has poor mechanical properties. The tensile strength and elongation of the
keratin–sodium alginate scaffold were intensively studied by Gupta and Hartrianti (Gupta and
Nayak 2016;Hartrianti et al., 2017). However, the introduction of other substances may reduce the
biocompatibility of the eventual scaffold.
Some studies have tried to make pure keratin scaffolds (Saul et al., 2011;Burnett et al., 2013;Wang
et al., 2015), but most of the preparation and research of hydrogels has focused on human hair.
However, the color of hair might vary considerably and contain a variety of chemical dye
Edited by:
Helan Xu,
Jiangnan University, China
Reviewed by:
Junheng Zhang,
South-Central University for
Nationalities, China
Bihe Yuan,
Wuhan University of Technology,
China
*Correspondence:
Zhiming Shi
shizm@imut.edu.cn
Specialty section:
This article was submitted to
Polymeric and Composite Materials,
a section of the journal
Frontiers in Materials
Received: 15 May 2021
Accepted: 19 July 2021
Published: 19 August 2021
Citation:
Wang X, Shi Z, Zhao L and Shen X
(2021) Low Concentration and High
Transparency Keratin Hydrogel
Fabricated via Cryoablation.
Front. Mater. 8:710175.
doi: 10.3389/fmats.2021.710175
Frontiers in Materials | www.frontiersin.org August 2021 | Volume 8 | Article 7101751
ORIGINAL RESEARCH
published: 19 August 2021
doi: 10.3389/fmats.2021.710175
composition; in addition, there are high concentration of keratin
solution and poor formability of pore. It is well known that the
efficiency of extracting keratin is low, which greatly limits the
industrial process of preparing hydrogels with keratin. Therefore,
it is particularly necessary to find suitable keratin resources and
prepare keratin gel with low concentration and high purity by
environmentally friendly methods.
As a simple and clean technique for preparing hydrogels,
cryogelation has been used more and more. The preparation of
keratin hydrogels by cryogelation is mainly based on the self-
assembly of keratin. The solvent crystallizes when the
temperature is below freezing point, and the keratin
macromolecules combine with each other by disulfide bonds,
hydrogen bonds, hydrophobicity, electrostatic attraction, and van
der Waals forces (Esparza, 2018b), when the temperature returns
to above freezing point, as the solvent crystals melt, solvent and
other components are enveloped in a three-dimensional network
of keratin, an interconnected microporous hydrogel structure is
formed (Lozinsky 2002;Henderson et al., 2013). In a recent study
by Chua et al. (Cui et al., 2019;Chua et al., 2020;Zhao et al., 2020),
HHK sponges was prepared by this method. However, as with
previous studies, the main research focuses on the mechanical
and biocompatibility of hydrogels, whether pure or hybrid keratin
hydrogels.
Herein, keratin extracted from rabbit hair with rich resources,
high amino acid content and poor spinnability was used as the
research object, a low concentration, high purity, and transparent
keratin hydrogel was prepared by simple freeze–thaw (FT) cycle.
It has been confirmed that the extracted rabbit hair keratin is
nontoxic. In this study, the feasibility of preparation of rabbit hair
keratin (RHK) hydrogel by cryogelation was studied. The
morphology and structure of RHK hydrogel were tested, and
the optical properties of keratin were comprehensively evaluated.
EXPERIMENTAL
Materials
Rabbit hairs were collected from German Angora rabbit warren
(Gansu, China). Urea and sodium bisulfite were purchased from
Damao chemical reagent Co. Ltd (Tianjin, China). Sodium
dodecyl sulfate (SDS) was purchased from Usolf Co. Ltd.
(Shenzhen, China).
Preparation of Rabbit Hair Keratin
RHK was extracted according to literature (Wang et al., 2021),
with modifications. Briefly, the rabbit hair defatted with
petroleum ether and anhydrous ethanol was subjected to
ultrasonic treatment. And then, it was immersed in mixed
urea-sodium bisulfite-sulfate solvent, and the solution was
heated and mechanically stirred for 4.5 h. The solution was
filtered to remove the undissolved rabbit hair, and the
obtained rabbit keratin solution was subsequently dialyzed in
distilled water using a dialysis tube for 48 h to remove small
molecules and salt formed in the reaction; during which, the
distilled water was changed every 4 h. The dialysis solution was
stored at 4°C for the following experiments.
Freeze–Thaw Cycles for Rabbit Hair Keratin
Hydrogel
Dialyzed solution with mass concentration of 2% was kept in a
refrigerated circulating device at −20°C for 12 h, thawed at 4°C,
and then the RHK hydrogel was prepared after several FT cycles.
The hydrogel was freeze-dried at −80°C to prepare keratin
scaffolds for morphology and structure testing, and the RHK
solution without the FT treatment was used as control.
Characterization and Measurements
Oscillatory rheology: The self-assembly gelation and the change of
rheological characteristics during reversible phase transition of
keratin hydrogels were characterized using a rheometer (Anton
Paar MCR92, Germany). RHK hydrogels were cast in 25 mm Petri
dishes. The oscillatory frequency sweep experiments were
performed at angular frequency of 0.1–100 rad/s at a constant
strain of 1% at 25°C, and the elastic modulus (G′) and viscous
modulus (G″) were recorded. The apparent viscosity change of
50 mL keratin solution during gelation was measured using a
rheometer (Brookfield R/S Plus Rheometer, United States) at 25°C.
Scanning electron microscopy (SEM): The RHK scaffold was cut
into thin slices with a sharp knife to expose clean cross sections.
Samples were gold-sputtered at 18 mA and observed using a
scanning electron microscope (650 FEG, FEI Quanta) at an
accelerating voltage of 20 kV under high vacuum. We
characterized the pore size within the hydrogel microarchitecture
from the SEM images using image processing techniques.
Porosity measurement: The porosity of the RHK scaffold was
tested using a liquid displacement method with absolute ethanol
(Loh and Choong, 2013). Briefly, put the RHK scaffold into a certain
volume (V
1
) of ethanol and record the volume (V
2
)ofthesolution
after 1 h. Next, the liquid-impregnated scaffold was removed, and
the remaining liquid volume (V
3
) was recorded. The porosity of the
RHK scaffold was calculated according to Equation (1) as follows:
porosity%V1−V3/V2−V3×100%.(1)
Fourier transform infrared spectroscopy: Chemical structures
of RHK hydrogels and RHK were characterized using a Fourier
transform infrared (IR Affinity-1 FTIR, Shimadzu) spectroscope
operated in the transmission mode. The FTIR spectra were
recorded at a wave number range of 4,000 cm
−1
to 400 cm
−1
,
at a resolution of 4.0, and at 40 scans per sample.
Raman spectroscopy: Raman spectra were obtained via a
Raman microscope (INVIA REFLEX03040404, Renishaw). The
laser excitation was provided with an argon ion laser operating at
10.2 mw of 633 nm output. Spectra were recorded between 2,000
and 300 cm
−1
. For each sample, about three replicates were
measured.
X-ray diffraction: Crystal structures of RHK hydrogels and
RHK were determined by X-ray diffractometer (D/MAX-2500/
PC XRD, Rigaku) operated using a Cu Kαradiation source. The
samples were scanned at a 2αBragg angle range of 5°–60°at 0.02°
step size and a scan speed of 3°/min.
Optical properties: The transmittance of RHK samples was
measured by UV-visible spectrophotometer (UV-2700,
Shimadzu). The scanning range is 300–800 nm. Endogenous
Frontiers in Materials | www.frontiersin.org August 2021 | Volume 8 | Article 7101752
Wang et al. Keratin Hydrogel Fabricated via Cryoablation
fluorescence spectra of proteins were determined at room
temperature using fluorescence spectrophotometer (G9800A,
Agilent). The excitation wavelength is 300 nm, and the
scanning range is 300–550 nm.
Measurement of particle size distribution: The particles size
distribution of RHK butadiene was determined by dynamic laser
scattering at 25°C.
Compressive measurement: Keratin hydrogels were prepared
into cylinders with a diameter of 10 mm and a height of 13 mm.
Uniaxial compression was conducted at RT using a universal
mechanical tester (Shimadzu, Japan) with 30 kN load cell. The
cross-head speed was set at 0.1 mm/min and the tests were
terminated.
RESULTS AND DISCUSSION
Rheological Properties of Rabbit Hair
Keratin Hydrogels
The gelation process of low concentration RHK solution was
illustrated in Figure 1A. The RHK solution self-assembled into
hydrogels without the adding of any chemical cross-linking agent
under FT cycles. The transparent and flowing RHK solution was
gradually converted to a semi-solid gel state through two FT
cycles. With the continuation of FT, RHK solution forms a single
highly transparent, intact piece of gel with a three-dimensional
network structure. The RHK hydrogel (FT3)broken by shearing
can form the initial gel state after being cultured at −20°C for 12 h
(Re-gel). From this knowable, RHK hydrogel is a reversible
thixotropic system. Noticeable feature is the absence of distinct
phase separation between the keratin hydrogels and the
surrounding aqueous medium as the article says (Zhao et al.,
2020). We believe that the formation of keratin hydrogels is due
to the three-dimensional network structure formed by the
binding of keratin molecules, bound water adsorbed by keratin
hydrophilic groups, and the unbound water clamped in the pore
structure.
In order to further elucidate the rheological properties of low-
concentration RHK solution during FT cycles, the apparent
viscosity, G′, and G″measurements were performed on the
RHK samples. In Figure 1B, comparative analysis between
RHK samples after different FT cycles shows that the apparent
FIGURE 1 | (A) Gelation and Reversible transformation of RHK, (B) viscosity and, (C) oscillatory amplitude sweep measurement of RHK samples at varied FT
cycles.
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Wang et al. Keratin Hydrogel Fabricated via Cryoablation
viscosity of RHK solution did not change and was almost zero,
this is consistent with the actual gelation phenomenon
(Figure 1A-FT1) after one FT cycle. The apparent viscosity of
the RHK hydrogels increased with the number of FT cycles after
FT2. For all samples, the apparent viscosity decreased with
increasing shear rates, suggesting a shear-thinning behavior
that is typical of viscoelastic fluids and gels, which could be
attributed of weak interactive forces or chain entanglements
between RHK at high shear rates.
G′is used to evaluate the strength or solid-like behavior of
hydrogels. It is related to the ability of material to store energy and
return to its original shape after being subjected to stress.
Assessment on the viscoelastic properties of hydrogels (FT3,
FT6, and FT9) and re-gel sample was conducted with
oscillatory rheology (Figure 1C). The experiments show that
the G′is significantly higher than G″, thereby suggesting that
these samples behave more like a viscoelastic gel rather than a
viscous fluid (Fu et al., 2020). Furthermore, G′increased with the
FT cycles, with the highest recorded value of 2,483.3 Pa in FT9
samples, which indicated that the rheological properties of the
hydrogels depended on the FT cycles. It is interesting that the G′
and G″of re-gel sample were higher than that of FT3 samples,
and it can be seen that the shear force cannot cause irreversible
rupture of the keratin hydrogels.
Rabbit Hair Keratin Scaffolds Morphology
Bioporous materials not only provide space for cell proliferation
and survival (Wang et al., 2012) but also provide a
microenvironment for the maintenance and release of bioactive
molecules (Chen and Mooney 2003). To further elucidate the
correlation between macroscopic behavior and micro
morphology, the morphologies of RHK scaffolds freeze-dried at
−80°C with different FT cycle were shown in both low- and high-
magnification SEM images (Figure 2A). The morphologies of
RHK solution sample freeze-dried at −80°C presented obvious
lamellae albeit structure, but the morphology of RHK scaffolds
appears to have regions of regular porous structure with
interconnected pores. Cui et al. (2019) prepared the hydrogel
with 20 mg/ml pure human hair keratin solution, which is the
relatively low concentration used for preparation of keratin
hydrogel at present. However, the microstructural imaging
shows that the porous structure of keratin hydrogel is incomplete.
Ge et al. (2021) prepared rabbit hair keratin with L-cysteine
hydrogel by the heating and cooling method, and the keratin
FIGURE 2 | (A) SEM images, (B) pore size distribution, and (C) porosity.
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Wang et al. Keratin Hydrogel Fabricated via Cryoablation
hydrogel showed an uneven porous structure. The shape of the
hole gradually changes from approximately round to rectangular
as FT cycles increases, and even to flake. At the same time, the
wall thickness of the hole also increases gradually. This
distinction in morphology is evident in FT3 and FT9 samples.
It can be inferred that the formation of columnar ice crystals
affects the shape of keratin hydrogel pores during FT processes.
The damaged hydrogels are still able to maintain intact porous
structures after the FT cycle.
The maximum distance of each hole was selected for the test
because the shape of hole of the FT9 scaffolds tends to be
rectangular. Figure 2B shows that the aperture of the three-
dimensional keratin scaffold increases with the FT cycles.
Apertures of the keratin scaffolds were 127 um, 232 um, and
324 um, respectively. Figure 2C shows that porosity of the keratin
scaffold is as high as 92%. In addition, FT cycles have a significant
effect on aperture, but had no obvious effects on the porosity of
keratin scaffolds. After vacuum freeze drying, the moisture
content directly affects the porosity because the keratin
hydrogels gradually dehydrate to form a sponge scaffold. As
can be seen from Figure 1, there is almost no water loss in
the process of FT cycles, so the volume of hydrogels had little
change after forming, which may be the reason why the FT cycles
have little influence on the porosity.
Structure of RHK Hydrogels
The FTIR spectra of RHK samples were observed in the range of
400–4,000 cm
−1
(Figure 3A), where they could be assigned to the
stretching vibrations of –N–H, which occurs in the range of
3,200–500 cm
−1
(Amide A), the stretching vibration CO which
fall at 1,654 cm
−1
(Amide I), the out-plane bending vibration of
N–H and C–H stretching which falls in 1,480–1,580 cm
−1
(Amide
II), and the in-phase stretching vibration of C–N which fall at
1,238 cm
−1
(Amide III) (Zhang, Zhao, and Yang 2015).
Functional groups and chemical bonds were generated after
the FT treatment of keratin solution. The amide I region of
the infrared spectrum of keratin is shown in Figure 3B. It can be
seen that the typical absorption peak at amide I region 1,654 cm
−1
of the infrared spectrum of keratin corresponds to the
intermolecular βfolding structure. After FT cycle, the position
of the absorption peak did not have any deviation. Compared
with amide I, amide II band is mainly sensitive to environmental
changes of the N–H group. Therefore, the amide II band can be
used to infer changes in the hydrogen bond microenvironment.
FIGURE 3 | (A–B) The FTIR spectra of RHK samples, (C) Roman spectra, and (D) XRD.
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Wang et al. Keratin Hydrogel Fabricated via Cryoablation
Belton studies have found that strong N–H hydrogen bond
groups are absorbed at high frequencies, that is, the stronger
the high wave number absorption, the more the hydrogen bond
groups are (Belton et al., 1995;Almutawah et al., 2007). As can be
seen from Figure 3B, compared with RHK solution, the
absorption peak of RHK hydrogels at 1,543 cm
−1
was
significantly higher than that at 1,523 cm
−1
, indicating that
hydrogen bond association in macromolecular peptide of
keratin was enhanced to varying degrees after FT. This is due
to the re-formation of hydrogen bonds between keratin molecules
during FT cycles which weakens the association between protein
molecules and water molecules.
The Raman spectra of RHK samples were observed in the
range of 400–1,800 cm
−1
(Figure 3C), where they could be
assigned to Amide I (1,597–1,680 cm
−1
), Amide III
(1,229–1,310 cm
−1
), and the S-S bond (506–550 cm
−1
)(Yu
2002); among them, Amide I and Amide III are very sensitive
to protein changes. The absorption peak of keratin solution at
1,684 cm
−1
corresponds to the intermolecular cfolding structure,
which is not present in keratin hydrogels. The peaks of the Amide
III are shifted, but all of them are within the range of
1,313–1,337 cm
−1
, which is a signal of β-rotation. Compared
with keratin solution, the peak of disulfide bond of RHK
hydrogel is wider, which may be related to the sulfhydryl
group being oxidized to disulfide bond in the transaction
process. Notable phenomenon is that the absorption peak of
amide Ⅵregion at 604 cm
−1
corresponds to the CO outside
curved surface signal becomes apparent in the RHK hydrogels.
In the XRD pattern in Figure 3D, the α-helix of RHK
hydrogels (2θ9.48°) shifted to the lower value compared
with RHK solution (2θ19.44°), especially in the FT6 and
FT9 samples. At the same time, the β-sheet of RHK hydrogels
(2θ20.06°) shifted to the higher value compared with RHK
solution (2θ19.44°). However, the variation of β-sheet of FT6
and FT9 samples was not as obvious as that of the FT3 sample.
This is perhaps because of the increase in the interactions between
keratin after several FT cycles. The interactions hindered the
α-helix to β-sheet conversion to some extent, while they provided
more chance for clustered random coils to form β-sheet.
Optical Properties of RHK Hydrogels
Figure 4A shows the result of light transmittance measurements
versus wavelength for the keratin before and after the gel. The
purified RHK solution offered high transmittance about 88% in
visible wavelengths, and the maximum transmittance of keratin
hydrogels prepared by FT can reach 67.57%. The transmittance
FIGURE 4 | (A) Optical transmittances of keratin hydrogel at varied FT cyc les, (B) photographs of the glass and keratin hydrogel, (C) DLS of keratin solution, and (D)
room temperature excitation and emission spectra.
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Wang et al. Keratin Hydrogel Fabricated via Cryoablation
test showed that the transparency of RHK decreased with the
increase in FT cycles. Furthermore, the optical transparency of
the RHK hydrogels was examined by comparing the appearance
of these hydrogels with that of glass substrate (Figure 4B). As
shown in the figure, the appearance of hydrogels formed after
three FT cycles is similar to that of glass, and the background
handwriting can still be seen through hydrogels after nine FT
cycles. This phenomenon is rarely reported in other keratin
hydrogels.
Pure wool keratin hydrogels concentration of 80 mg/ml
prepared by al.(Chen et al., 2021) at room temperature
showed milky white, while pure human hair keratin hydrogels
of 20 mg/ml prepared by Cui et al.(Cui et al., 2019)at−20°C
showed yellowish white. Keratin hydrogels are used as medical
adjuvant, and its high transparency is conducive to observing the
healing of wounds.
The high transparency of RHK hydrogels is not only related to
the low concentration of 2% keratin but also related to the degree
of cross-linking of macromolecular chains and the uniformity of
pore size distribution in the process of gel formation (Chen 2002).
During the FT process, the three-dimensional network structure
of RHK hydrogels is formed through the cross-linking of keratin
macromolecules, and there is liquid water, uncross-linked keratin
molecules, and cross-linked regions in the hydrogels. The
existence of liquid water would not affect the transparency,
and the uncross-linked keratin molecules will have a certain
influence on the transparency of the hydrogel (Hassan and
Peppas 2000). Therefore, the particle size of keratin was
measured as shown in Figure 4C. RHK particle size
distribution curve [G(d)] results show that the particle size of
keratin mainly concentrates between 25 and −150 nm, and the
particle size accumulation curve [C(d)] data show that the
number percentage of particle size less than 100 nm is more
than 82.5%. The particle size was less than half of the incident
light, and the substance formed was relatively transparent. It can
be seen that the small particle size and narrow distribution range
of keratin may also be one of the reasons for the formation of
transparent hydrogels. The transmittance of RHK hydrogels
decreases with the increase in FT cycles, which may be due to
the high crystallinity and surface roughness caused by the
aggregation and cross-linking of keratin molecules. At the
same time, SEM results show that the pore size is greatly
different, and the distribution of keratin hydrogels is uneven
with the increase in FT times, which will cause serious light
scattering and thus affect the transparency.
Fluorescent biomaterials have always received attention
because they can be tracked in vivo and needs no external
fluorophore. Keratin is a biological material with natural
fluorescent properties because it contains residues of
tryptophan, tyrosine, and phenylalanine, which absorb
ultraviolet light and emit fluorescence. To study whether it
still had the inherent protein fluorescence properties, we
examined the emission spectra of the keratin hydrogels.
Figure 4D shows the emission spectra of RHK solution and
hydrogels. When monitored at 354 nm, the obtained emission
spectrum of keratin solution consisted of one broad band in the
wavelength range of 280–320 nm with a peak at 300 nm. The
resulting emission spectrum was composed of a broad emission
band in the wavelength range from 300 to 500 nm, and emission
peak was found to be 354 nm, which was close to fluorescence
spectra of tryptophan (Burstein et al., 2001). Besides, the stoke
shift and full width at half maxima (FWHM) of the emission band
were calculated as 54 and 67 nm, respectively.
The inherent fluorescence wavelength and fluorescence
intensity of protein are affected by the tertiary structure of
protein, the spatial distribution of fluorescent amino acids in
protein, the interaction of side chain groups with other residues
or solvents, and the energy resonance transfer within fluorescent
amino acids (Ross et al., 1997). Compared with RHK solution, the
peak generates bathochromic shift in the emission spectrum of
hydrogels and the fluorescence intensity is reduced. This may be
because disulfide bond, hydrogen bond, ionic bond, and
hydrophobic bond are formed in the gelation process of
keratin solution, which strengthens the interaction between
proteins and reduces the amount of charge on the surface of
protein molecules, leading to the gradual exposure of the side
chain groups of fluorescent amino acid molecules to the aqueous
solution. At this time, the polarity of the environment in which
the chromogenic amino acid is located gradually increases, the
extension degree of the peptide chain increases, and the content
of helical structure decreases. This was also confirmed by the
structural test results of keratin hydrogels.
The transmittance test showed that the transparency of
keratin hydrogels decreased with the increase in FT cycles.
The fluorescent biomaterial test results also showed that the
more the FT cycles of keratin hydrogels, the smaller the red shift
FIGURE 5 | Compression process of RHK scaffolds.
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Wang et al. Keratin Hydrogel Fabricated via Cryoablation
of the emission spectrum peak and the lower the fluorescence
intensity. This is also related to the fact that a small amount of
water will be removed from the keratin hydrogels after multiple
FT cycles.
Compressive Properties
The compressive properties of the RHK hydrogel are presented in
Figure 5 and Figure 6. From Figure 5, the deformation of keratin
hydrogel increases continuously under the action of pressure, the
hydrogel did not break after the removal of external force when
the deformation reached more than 80%, and the deformation of
the hydrogel recovers. The reduced volume of keratin hydrogel
after deformation recovery is due to the removal of partial
unbound water under external force. It can be seen that the
keratin hydrogel has good elasticity. The compressive strength
was also significantly increased with more freeze–thaw cycles
(Figure 6). The compressive stress of RHK hydrogel (FT9)
reached to 14.927 kPa when the compressive strain is 53%.
CONCLUSION
In a nutshell, low concentration and highly pure RHK hydrogels
were fabricated by green and simple freeze–thaw technique
without adding extraneous reagents. The morphology,
structure, and physical properties of RHK hydrogels can be
adjusted by controlling the FT processing parameters. The
RHK hydrogels shows unique optical properties, and its
transparency is up to 67.57%, while maintaining its
fluorescence properties, which is conducive to the observation
of wound healing and location of drug delivery.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/supplementary material, further inquiries can be
directed to the corresponding author.
AUTHOR CONTRIBUTIONS
XW: data curation and writing—original draft. ZS: methodology,
supervision, review and editing. LZ and XS: investigation; All
authors contributed to the article and approved the submitted
version.
FUNDING
This research was supported by Natural Science Foundation of
Inner Mongolia (2020LH05005), and the Foundation of Inner
Mongolia University of Technology (ZZ201817).
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